Fucose is found widely distributed in the complex carbohydrates and glycoconjugates of bacteria, plants, and animals. In these organisms it plays diverse roles, ranging from its involvement in nodulation in Azorhizobium [1] to development of shoots in Arabidopsis [2] to adhesion of leukocytes to activated endothelia in humans as part of the selectin ligand [3]. In humans a defect in GDP-fucose biosynthesis is responsible for the immune disorder Leukocyte Adhesion Deficiency type II [4, 5, 6]. Fucose is added to these glycoconjugates by specific transferases that use GDP-fucose as the sugar donor. GDP-fucose in turn is synthesized primarily from GDP-mannose in a three-step reaction involving two enzymes as shown in FIG. 1. The first step is the oxidation at C4 of the mannose ring and subsequent reduction at C6. This is carried out by a NADP+ dependent enzyme, GDP-mannose 4,6 dehydratase (GMD) [7, 8, 9]. The next two steps of the reaction, the epimerization at C3 and C5 of the mannose ring and the subsequent NADPH dependent reduction at C4 to yield GDP-fucose, are carried out by a single dual function enzyme, GDP-fucose synthetase (GFS) [9, 10, 11]. In E. coli this enzyme is encoded by the fcl gene, previously known as wcaG [12, 13]. It is in these final two steps that GDP-fucose biosynthesis differs from synthesis of other deoxy sugars derived from dTDP-glucose and CDP-glucose. In the latter pathways, separate epimerase and reductase enzymes encoded by independent genes perform the roles of the dual function epimerase-reductase of the GDP-fucose pathway (reviewed in [14]).
The human homologue of GFS has recently been identified as the FX protein [11]. As with the E. coli enzyme it is a homodimer that binds NADP(H) and catalyzes both the epimerization and reduction of GDP-4-keto, 6-deoxy-mannose. Human GFS has 29% identity to the E. coli protein. More distantly related to both the human and E. coli enzymes is UDP-galactose-4-epimerase (GalE), which catalyzes the reversible epimerization of UDP-glucose to UDP-galactose. Essential to catalysis is a tightly bound NAD+ that is reduced and then oxidized during the catalytic cycle. UDP-galactose 4-epimerase is a member of the short chain family of dehydrogenase/reductases (SDR) (reviewed in [15]). This family of enzymes catalyzes a diverse set of enzymatic reactions spanning 5 E.C. classes using a conserved set of active site residues including a Ser-Tyr-Lys catalytic triad.
It would, therefore, be desirable to determine the structure of E. coli GDP-fucose synthetase in order to facilitate the identification and development of agonists and antagonists of GFS enzyme activity in humans and other species.
We have determined the structure of GDP-fucose synthetase from E. coli at 2.2 xc3x85 resolution. The structure of GDP-fucose synthetase is closely related to that of UDP-galactose 4-epimerase and more distantly to other members of the short chain dehydrogenase/reductase family. We have also determined the structures of the binary complexes of GDP-fucose synthetase with its substrate NADPH and its product NADP+. The nicotinamide cofactors bind in the syn or anti conformations, respectively.
GDP-fucose synthetase binds its substrate, NADPH, in the proper orientation (syn) to transfer the pro-S hydride. We have observed a single binding site in GDP-fucose synthetase for the second substrate, GDP-4-keto, 6-deoxy-mannose. This implies that both the epimerization and reduction reactions occur at the same site on the enzyme. As for all members of the short-chain family of dehydrogenase/reductases, GDP-fucose synthetase retains the Ser-Tyr-Lys catalytic triad. We propose that this catalytic triad functions in a mechanistically equivalent manner in both the epimerization and reduction reactions. Additionally, the x-ray structure has allowed us to identify other residues potentially substrate binding and catalysis.
The present invention provides for crystalline GFS. Preferably, the GFS is E. coli GFS, although GFS from other species are also included within the invention. In certain embodiments, the GFS is recombinant GFS and/or comprises the mature sequence of naturally-occurring GFS.
Other embodiments provide for a crystalline composition comprising GFS in association with a second chemical species. Preferably, the second chemical species is selected from the group consisting of NADPH, NADP+ and a potential inhibitor of GFS activity.
Yet other embodiments provide for a model the structure of GFS comprising a data set embodying the structure of GFS. Preferably, such data set was determined by crystallographic analysis of GFS, including possibly by NMR analysis. In certain embodiments, the data set embodies a portion of the structure of GFS, including without limitation the active site of GFS.
Any available method may be used to construct such model from the crystallographic and/or NMR data disclosed herein or obtained from independent analysis of crystalline GFS. Such a model can be constructed from available analytical data points using known software packages such as HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS, QUANTA, BUSTER, SOLVE, O, FRODO, RASMOL, and CHAIN. The model constructed from these data can then be visualized using available systems, including, for example, Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM, and Compaq. The present invention also provides for a computer system which comprises the model of the invention and hardware used for construction, processing and/or visualization of the model of the invention.
Further embodiments provide a computer system comprising computer hardware and the model of the present invention.
Methods are also provided for identifying a species which is an agonist or antagonist of GFS activity or binding comprising: (a) providing the model of the present invention, (b) studying the interaction of candidate species with such model, and (c) selecting a species which is predicted to act as said agonist or antagonist. Species identified in accordance with such methods are also provided.
Other embodiments provide: (1) a process of identifying a substance that inhibits GFS activity or binding comprising determining the interaction between a candidate substance and a model of the structure of GFS, or (2) a process of identifying a substance that mimics GFS activity or binding comprising determining the interaction between a candidate substance and a model of the structure of GFS. Substances identified in accordance with such processes are also provided.
The study of the interaction of the candidate species with the model can be performed using available software platforms, including QUANTA, RASMOL, O, CHAIN, FRODO, INSIGHT, DOCK, MCSS/HOOK, CHARMM, LEAPFROG, CAVEAT(UC Berkley), CAVEAT(MSI), MODELLER, CATALYST, and ISIS.
Other embodiments provide a method of identifying inhibitors of GFS activity by rational drug design comprising: (a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the GFS sequence based upon the crystal structure co-ordinates of GFS; (b) synthesizing the inhibitor; and (c) determining whether the potential inhibitor inhibits the activity of GFS. In other preferred embodiments, the inhibitor is designed to interact with one or more amino acids selected from the group consisting of Arg12, Met14, Val15, Arg36, Asn40, Leu41, Ala63, Ile86, Gly106, Ser107, Ser108, Cys109, Tyr136, Lys140, Asn165, Leu166, His179, Val180, Leu184, Val201, Trp202, Arg209, and Lys283.
Agonists and antagonists identified by such methods are also provided.
A process is also provided of identifying a substance that inhibits human FX protein activity or binding comprising determining the interaction between a candidate substance and a model of the structure of GFS of the present invention.
Other embodiments provide for a method of identifying inhibitors of human FX protein activity by rational drug design comprising:
(a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the GFS sequence based upon the crystal structure co-ordinates of crystalline GFS of the present invention;
(b) synthesizing the inhibitor; and
(c) determining whether the potential inhibitor inhibits the activity of human FX protein.
In preferred embodiments, the inhibitor is designed to interact with one or more amino acids in the GFS sequence selected from the group consisting of Arg12, Met14, Val15, Arg36, Asn40, Leu41, Ala63, Ile86, Gly106, Ser107, Ser108, Cys109, Tyr136, Lys140, Asn165, Leu166, His179, Val180, Leu184, Val201, Trp202, Arg209, and Lys283.
Agonists and antagonists identified by such methods are also provided.